Project Summary Microglia, CNS-resident macrophages, get activated by pathogens and endogenous damage signals through cell surface receptors including toll like receptors (TLRs) and TNF? receptors and thereby mediate inflammatory responses including upregulation of inflammatory gene expression and generating inflammatory prostaglandins. These mediators directly cause neurotoxicity, which in turn further activates microglia, initiating a self- propagating cycle of chronic neuroinflammation. This phenomenon is directly implicated in the pathogenesis and progression of multiple chronic neurological diseases, including Parkinson?s disease, Alzheimer?s disease and neuropathic pain. These diseases also have in common a lack of effective, disease modifying therapies, resulting in chronic and relapsing disease. Thus, there is an urgent unmet medical need to identify novel therapeutic targets and lead compounds to improve treatment options and prognosis. Targeting dysregulated inflammatory signaling in microglia is a promising therapeutic strategy; however, strategies regulating individual inflammatory signaling pathways have lacked clinical efficacy. The long-term goal of the proposed studies is to target multiple inflammatory pathways, which would be more clinically efficacious, and possibly disease modifying, in CNS diseases with underlying chronic neuroinflammation. The rationale for this proposal is that the microglial protein Regulator of G protein Signaling 10 (RGS10) inhibits pro-inflammatory signaling downstream of TLR4 and TNF? receptors, and protects against inflammation-induced neurotoxicity. RGS10 expression is strongly suppressed through epigenetic mechanisms following microglial activation and this silencing amplifies microglial signaling in a feed-forward mechanism that contributes to dysregulation of inflammatory signaling and contributes to chronic neuroinflammation. The central hypothesis is that blocking the suppression of RGS10 expression in activated microglia will diminish multiple inflammatory pathways and limit neuroinflammation. The overall objective of this proposal is to develop a non-biased high-throughput screening (HTS) assay to identify and validate small molecule regulators of RGS10 expression in resting and activated microglia through three aims. 1) Develop a stable microglial cell line expressing endogenous RGS10 tagged with Nanoluc luciferase, using CRISPR/Cas9 technology, and optimize a Nano-Glo luciferase assay. 2) Perform HTS of 30,000 small molecules for their ability to a) block TLR4-mediated RGS10 suppression or b) enhance basal RGS10 levels in microglia. 3) Validate identified hits to identify the most promising candidates for further development. The proposed approach is innovative because it targets regulation of endogenous RGS10 expression under physiologically relevant conditions. In addition, targeting epigenetic regulation of RGS10 is a highly novel approach to control key inflammatory signaling pathways in microglia. Furthermore, suppression of RGS10 expression also occurs in multiple systems (macrophages, cardiac myocytes, and ovarian cancer). Thus, this research is significant because it introduces a novel approach to modulate a novel target with broad therapeutic implications.